Chemical reaction monitor

ABSTRACT

Disclosed herein are systems for monitoring chemical reactions. The systems can comprise a lighting device, a camera device for obtaining an image of the chemical reaction mixtures and an analyzer program to process data obtained from the image. Also disclosed are methods of monitoring the progress of chemical reactions using these systems.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/762,931 filed Jan. 21, 2004, now U.S. Pat. No. 7,887,752. Thisapplication claims priority to U.S. Provisional Patent Application No.60/441,752 filed Jan. 21, 2003, entitled CHEMICAL REACTION MONITOR, theentire contents of the foregoing applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of monitoring chemicalreactions. More particularly, the present invention relates to a new andimproved chemical reaction monitor for use in monitoring parallelchemical reactions.

The chemical reaction monitor of the present invention is particularlysuited for real-time monitoring of oligonucleotide synthesis. Thechemical reaction monitor of the present invention is also particularlysuited for providing a quality control (QC) measure for oligonucleotideproduction.

2. Description of the Related Art

Oligonucleotide synthesis is a cyclical process that assembles a chainof nucleotides. Nucleotides are added one by one through a cycle ofchemical reactions, in which a particular molecule (e.g., a nucleotide)is added to a growing DNA molecule (e.g., a growing DNA chain),sometimes via catalysis, until the desired chain is complete. Generally,each cycle of chemical reactions includes the steps of detritylation,coupling, capping and oxidation.

During the detritylation or “deprotection” step, a dimethoxytrityl (DMT)group is removed from the last nucleotide of the growing DNA chain toallow the addition of the next nucleotide. The amount of DMT releasedfrom each cycle is monitored to insure a high coupling efficiency. Therelease of DMT is apparent because a bright orange color is emitted asDMT is released.

Monitoring of the detritylation or deprotection step in known commercialsynthesizers is done in the form of a spectrophotometer monitoring acuvette through which wash waste is passed. Such monitoring is done on adiscrete per sample basis in systems that process as many as 32oligonucleotide samples simultaneously. During the detritylation stepthe waste is monitored for the presence and magnitude of the orangecolor indicating the release of DMT. As the number of simultaneousreactions increases, the ability to use known methods to monitorreaction progress becomes prohibitively inefficient.

What is needed is a chemical reaction monitor that allows parallelmonitoring of chemical reactions in which each sample may be observedand monitored both discretely and as a collection. The present inventionsatisfies this need and provides other advantages as well.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a chemical reactionmonitoring system for parallel monitoring of a plurality of chemicalreactions wherein each chemical reaction occurs within a respective oneof a plurality of wells. The system includes a lighting device forilluminating the plurality of wells, a camera device configured toobtain a fresh image of the plurality of wells and saving the freshimage to an image storage location, a viewer program for viewing thesaved fresh image, and an analyzer program for accessing the saved freshimage, geometrically registering the saved fresh image in order todetermine a specific value corresponding to each one of the chemicalreactions within each well at the time the image was obtained, andsaving the specific values to an analysis results storage location.

A system of the invention can further include optics for directing lightfrom a lighting device to the sample and for directing the light fromthe sample to the camera.

In one embodiment, the chemical reaction is oligonucleotide synthesis.Thus, the device can be integrated into an oligonucleotide synthesisinstrument. The plurality of wells may be located in a multi-well plate.

The lighting device may include a light emitting diode (LED) array. TheLED array may have a first array and a second array positioned on eitherside of a multi-well plate-viewing window. Alternatively, the LED arraycan include a single array positioned pivotally mounted on one side of amulti-well plate-viewing window.

In one embodiment, the camera device is a charge couple device (CCO)capable of imaging the plurality of wells simultaneously: The cameradevice can be coupled with imaging optics for simultaneous imaging of aplurality of wells. The analyzer program may process the fresh image andsave a corresponding processed image to the analysis results storagelocation.

The invention further provides a chemical synthesis system. The systemmay include one or more of the following components: (a) a sample holderplaced to support a plurality of wells; (b) a liquid dispenser placed todispense a liquid sample to the plurality of wells; (c) a liquid removaldevice placed to remove the liquid sample from the plurality of wells;(d) an automated device for controlling said liquid dispenser or saidliquid removal device; (e) a lighting device for illuminating theplurality of wells; (f) a camera device configured to obtain a freshimage of the plurality of wells and save the fresh image to an imagestorage location; (g) an computer system configured to: (i) access thesaved fresh image; (ii) determine a specific value corresponding to eachone of the chemical reactions within each well at the time the image wasobtained; and (iii) communicate an instruction to the automated device.

Also provided is a chemical synthesis system that may include one ormore of the following components: (a) a sample holder placed to supporta plurality of wells; (b) a liquid dispenser placed to dispense a liquidsample to the plurality of wells; (c) a liquid removal device placed toremove the liquid sample from the plurality of wells; (d) a lightingdevice for illuminating the plurality of wells; (e) a camera deviceconfigured to obtain a fresh image of the plurality of wells and savethe fresh image to an image storage location; and (f) a computer systemconfigured to: (i) access the saved fresh image; (ii) determine aspecific value corresponding to each one of the chemical reactionswithin each well at the time the image was obtained; and (iii)communicate a representation of the specific values to a graphical userinterface.

In particular embodiments a liquid dispenser can be placed toindependently dispense various liquid samples to the plurality of wells.Thus, different samples can be added to each well. A liquid dispensercan be placed under computer control. Accordingly, commands can be sentfrom the computer directing the liquid dispenser to dispense liquid,stop dispensing liquid, dispense a particular volume of liquid, or todispense to a particular well in a plurality of wells being monitored.

Another aspect of the present invention is directed to a method forparallel monitoring of a plurality of chemical reactions, wherein eachchemical reaction occurs within a respective one of a plurality ofwells. The method includes the steps of illuminating the plurality ofwells, and obtaining images of the plurality of wells with a cameradevice and saving the images to an image storage location, the cameradevice being capable of imaging the plurality of wells simultaneously.

The method may further include the step of viewing one or more of thesaved images. The method may further include the step of analyzing thesaved images by opening each saved image and geometrically registeringeach saved image in order to determine a specific value corresponding toeach one of the chemical reactions at the time the image was obtained,and saving the specific values to an analysis results storage location.

The invention further provides a method for synthesizing a plurality ofdifferent polymers. The method may include one or more of the followingsteps: (a) providing a plurality of wells containing support-boundmonomeric or oligomeric precursors of the polymers; (b) dispensingsecond monomeric precursors of the polymers to the plurality of wellsunder conditions for forming an intermediate in which the support-boundmonomeric or oligomeric precursors are bound to the second monomericprecursors; (c) obtaining a fresh image of the plurality of wells andsaving the fresh image to an image storage location; (d) executingcommands in a computer system to access the saved fresh image,geometrically register the saved fresh image in order to determine aspecific value corresponding to each one of the chemical reactionswithin each well at the time the image was obtained, and save thespecific values to an analysis results storage location; and (e) if thespecific values are within a pre-defined passing range then repeatingsteps (a) through (d) and if the specific values are within apre-defined failing range then preventing repetition of steps (a)through (d) for at least one of the wells in the plurality of wells.

An object of the present invention is to provide a method and apparatusfor monitoring parallel chemical reactions in which each one of aplurality of samples may be observed and monitored both discretely andas a collection.

Another object of the present invention is to provide a method of usinga camera to allow each one of a plurality of samples to be observed andmonitored both discretely and as a collection.

Another object of the present invention is to provide a method of usinga single or collection of optical filtering, remote triggering of thecamera, remote triggering of an array of photodiodes, other suitableprocesses, or a combination thereof to allow a measurement of photons ofa specific spectral quality using the monitoring apparatus.

The chemical reaction monitor of the present invention has otherfeatures and advantages which will be apparent from or are set forth inmore detail in the accompanying drawings, which are incorporated in andform a part of this specification, and the following DetailedDescription of the Invention, which together serve to explain theprinciples of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a chemical reaction monitor of thepresent invention including a camera device and a lighting devicemounted on an oligonucleotide synthesizer for capturing images of amulti-well plate.

FIG. 2 is an enlarged perspective view of the lighting device of FIG. 1.

FIG. 3 is a schematic view of a chemical reaction monitor in accordancewith the present invention utilizing the chemical reaction monitor ofFIG. 1.

FIG. 4 is a graphical representation of DMT measurements of a well ofthe multi-well plate taken during an exemplary successfuloligonucleotide synthesis.

FIG. 5 is a graphical representation of DMT measurements of a well ofthe multi-well plate taken during an exemplary unsuccessfuloligonucleotide synthesis.

FIG. 6 is a schematic representation of a graphical user interface ofthe chemical monitor of FIG. 3 illustrating stored data regarding thestatus of a specified multi-well plate during a specified process.

FIG. 7 is a schematic representation of a graphical user interface ofthe chemical monitor of FIG. 3 providing an overview of a plurality ofnetworked oligonucleotide synthesizers.

FIG. 8 is a graph of an exemplary exponential decay showing yields as afunction of “cycle” based on a fictitious substance and couplingefficiency.

FIG. 9 is a graphical representation of DMT measurements of a well ofthe multi-well plate taken during an exemplary successfuloligonucleotide synthesis as compared to an expected DMT value.

FIG. 10 is a perspective view of another chemical reaction monitor ofthe present invention including a camera device and a-lighting devicemounted on an oligonucleotide synthesizer for capturing images of amulti-well plate.

FIG. 11 is an enlarged perspective view of the lighting device of FIG.10.

FIG. 12 is a perspective view of a chemical reaction monitor of thepresent invention including a camera device and a lighting devicemounted on an oligonucleotide synthesizer for capturing images of amulti-well plate.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the preferred embodiments of thepresent invention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications andequivalents, which may be included within the spirit and scope of theinvention as defined by the appended claims.

The chemical reaction monitor of the present invention is configured foruse in monitoring parallel chemical reactions and is particularly suitedfor realtime monitoring of polymer synthesis such as oligonucleotidesynthesis or peptide synthesis or both. The chemical reaction monitor ofthe present invention is also particularly suited for providing aquality control (QC) measure for oligonucleotide or peptide production.In one embodiment, the chemical reaction monitor is an automated DMTmonitoring system used to track the yield, quality and general state ofoligonucleotides (DNA) being synthesized at anyone time. Although thepresent invention is exemplified herein in the context of polynucleotidesynthesis one should appreciate that the chemical reaction monitor ofthe present invention is not limited to oligonucleotide synthesis andmay be used in the monitoring of other types of chemical reactions.

A chemical reaction monitor of the present invention can be used tomonitor any reaction that produces an optical signal indicative ofreaction progress. Accordingly, an optical signal that results directlyfrom a reaction can be monitored. For example as set forth below, therelease of optically inactive DMT during deprotection steps of apolynucleotide synthesis can be monitored. In particular embodiments, areagent can be added to a reaction to react with a product of thereaction thereby changing an optical property of the reaction mixture.For example, a reagent that becomes optically active in the presence ofa desired product, modifies a particular product to make the productoptically active, reduces optical activity of a desired product, orbecomes less optically active in the presence of a desired product canbe used. In particular embodiments, polypeptide synthesis can bemonitored based on the addition of a monitoring reagent such asbromophenol blue. Methods for peptide synthesis utilizing bromophenolblue to monitor the status of peptide coupling are known as described,for example, in U.S. Pat. No. 5,342,585, entitled APPARATUS FOR MAKINGMULTIPLE SYNTHESIS OF PEPTIDES ON SOLID SUPPORT and issued to Lebl etal. on Aug. 30, 1994. Other reagents that can be used in a polypeptidesynthesis and used in an apparatus of the invention for simultaneouslymonitoring a plurality of reactions include, for example,trinitrobenzene sulfonic acid which can be added to a polypeptidereaction or ninhydrin which can be added to an aliquot removed from apeptide reaction in accordance with the Kaiser test kit (FlukaChemicals, Cat No. 60017).

Any of a variety of known optical properties can be monitored inaccordance with the invention including, without limitation, absorbance,fluorescence, chemiluminescence, polarization, circular dichroism,fluorescence resonance energy transfer (FRET), light scattering or thelike. Furthermore, those skilled in the art will readily recognize thatan optical detection device, such as those set forth herein can bemodified or replaced with any of a variety of known detection devicesappropriate for monitoring such optical properties. Further still, theinvention can be used to detect changes in optical properties that occurin one or more range of the electromagnetic spectrum including, withoutlimitation, infrared, visible, ultraviolet, x-ray, microwave,sub-regions thereof such as red, blue or yellow sub-regions of thevisible region, combinations of these regions or subregions, or otherregions of the spectrum.

In accordance with the present invention, an oligonucleotide synthesizeris configured to acquire an image of a plurality of wells, such as thewells of a multiwell plate. As used herein the term “multi-well plate”is intended to mean a substrate having a plurality of discrete chamberssuitable for holding a liquid. A substrate included in the term can be,for example, molded plastic such as polystyrene or polypropylene.Exemplary multi-well plates include, for example, microplates,microtiter plates or n-well plates where “n” is the number of wellsincluding, for example, 8-, 16-, 96-, 384-, or 1536-wells. As usedherein, the term “microplate” is intended to mean a multi-well platethat has dimensions and properties consistent with the definitionprovided by the Society for Biomolecular Screening (Danbury, Conn.,USA). A multi-well plate can have wells with any of a variety ofcross-sectional shapes including, for example, cylindrical, square,rectangular, multisided, interlocking shapes wherein the bottom of wellsare flat, conical, pointed, or round.

A plurality of wells imaged or processed in accordance with thisdisclosure can be located in a single substrate or in multiplesubstrates. In one embodiment each well can be a separate tube occurringin a plurality of tubes. Such tubes can occur at fixed locations in aholder or substrate or can be moveable such that one or more of thetubes changes relative location with respect to another tube in theplurality.

The apparatus and methods set forth herein can be used to monitor largepluralities of reactions simultaneously including, for example, at least96, 100, 384, 500, 1000, 1536, 2000, 3000, 4000, 5000, 10,000, 100,000or more reactions. The reactions can occur simultaneously such that allare in the same stage of a reaction cycle or, alternatively, reactionsproceeding through different stages of a reaction cycle can bemonitored. Furthermore, different types of reactions can besimultaneously monitored. For example, an apparatus or method of theinvention can be used to simultaneously monitor synthesis of a pluralityof peptides and a plurality of polynucleotides.

An apparatus of the invention can be configured to acquire an imageduring any step of synthesis including, but not limited to, wash cycles,coupling cycles, capping cycles and oxidation cycles. One willappreciate that image acquisition may be useful in debugging machine orsoftware issues and for other technical means. Image acquisition isparticularly suited for obtaining images of the wells of a multi-wellplate during the deprotection step in order to determine DMT intensity.

Generally, an image is acquired, uniquely named and sent as a “fresh” orunprocessed image file to an image storage location. Upon arrival at thestorage location, an analyzer program can process the fresh image file.For example, upon detecting a fresh image, the analyzer program can openthe image, register the relative position of the multi-well plate tomitigate positional errors, and extract an optical intensity value fromeach well of the multi-well plate. Upon completion, the analyzer programcan write the optical intensity data to an analysis results storagelocation. The process image file may be renamed to indicate that theimage has been processed. In accordance with the present invention, thisprocess may take place in as little as approximately 8 seconds thusallowing the analyzer program to populate the analysis results databasewith near real-time data. An apparatus or method of the inventiongenerally includes a computer system such as those based on INTEL®microprocessors and running MICROSOFT WINDOWS® operating systems. Othersystems such as those using the UNIX® or LINUX®operating system andbased on IBM®, DEC® or MOTOROLA® microprocessors are also contemplated.The systems and methods described herein can also be implemented to runon client-server systems and wide-area networks such as the Internet.

As used herein the term “storage location” is intended to mean acomputer readable memory. Exemplary forms of computer readable memoryinclude, but are not limited to, a database, hard disk, floppy disc,compact disc, magnetooptical disc, Random Access Memory. Read OnlyMemory or Flash Memory. The memory or computer readable medium used inthe invention can be contained within a single computer or distributedin a network. A network can be any of a number of conventional networksystems known in the art such as a local area network (LAN) or a widearea network (WAN). Client-server environments, database servers andnetworks that can be used in the invention are well known in the art.For example, the database server can run on an operating system such asUNIX®, running a relational database management system, a World Wide Webapplication or a World Wide Web server. Other types of memories andcomputer readable media are also contemplated to function within thescope of the invention.

Data stored at a storage location can be in any of a variety of formsknown in the art including, for example, a text file, .xml file, .jpgfile, TIFF file, or BMP file. Software to implement a method of theinvention can be written in any well known computer language, such asJava, C. C++, Visual Basic, FORTRAN or COBOL and compiled using anywell-known compatible compiler. The software of the invention normallyruns from instructions stored in a memory on a host computer system. Adatabase or data structure of the invention can be represented in amarkup language format including, for example, standard generalizedmarkup language (SGML), hypertext markup language (HTML) or extensiblemarkup language (XML).

A computer system useful in the invention can further include alaboratory management system (LIMS). A LIMS system can containinformation relating to the sequence of a polymer to be synthesized asinformation about manipulations that have been and are to be carried outon the polymer. The entire sequence information regarding the synthesisof a polymer can be copied from LIMS into the analysis results storagelocation for quick, native retrieval by a technician or other authorizedpersonnel. This can provide the advantage of minimizing the calls toLIMS to just one per synthesis.

The chemical reaction monitor of the present invention generallyincludes a hardware component and a software component. The hardwarecomponent captures images of a plurality of wells of a multi-well platecontaining samples in which chemical reactions occur. The softwarecomponent can be subdivided into a storage subcomponent, an analyzersubcomponent, an analysis results and a viewing subcomponent.

In one embodiment shown in FIG. 1, a chemical reaction monitor 30includes a hardware component mounted on an oligonucleotide synthesizersystem 31 of the type including, but not limited to the OLIGATOR® DNAsynthesis platform, developed by Illumina, Inc. of San Diego, Calif.Similarly, the hardware component may be mounted on oligonucleotidesynthesizers such as, but not limited to, those disclosed by U.S. patentapplication Ser. No. 09/881,052, filed Jun. 13, 2001, entitledOLIGONUCLEOTIDE SYNTHESIZER, and now U.S. Pat. No. 6,663,832, the entirecontents of which is incorporated herein by this reference. Otherpolymer synthesizers useful in the invention are described in U.S. Pat.Nos. 5,338,831 and 6,121,054 and WO 00/44491, the entire contents ofwhich patents are incorporated herein by this reference. Reagents can bedispensed to wells and removed by centrifugation as described in theabove patent and patent application. Polymer precursors including, butnot limited to, monomeric and oligomeric precursors can be attached towells or other solid phase supports using methods known in the art asdescribed for example in the above patent and patent application.

One should appreciate that the hardware component or the softwarecomponents of the present invention or both may be used in combinationwith other systems in which multiple chemical reactions occur inparallel. Thus, reagents can be dispensed, manipulated, and removed fromwells using methods utilized in synthesizers from Applied Biosystems,Perkin Elmer and other commercial vendors. Furthermore, several chemicalsynthesizers, each having appropriate hardware components such as thoseset forth herein, can be networked for communication with a centralizedsoftware component such that multiple instruments can be monitored.

Chemical reaction monitor 30 can include a hardware component having acomputer accessible camera device 32, a lighting device 33 and a clientserver based software component for controlling the camera device and/orthe lighting device. The camera device and the lighting devicefacilitate capturing an image of a multi-well plate 34, including aplurality of wells 35 located therein, during specific points of achemical synthesis process.

The lighting device is positioned in a manner to illuminate a multi-wellplate of a synthesizer apparatus and the wells thereof, for example, asshown in FIG. 2 for an oligonucleotide synthesizer. Any lighting sourcethat allows wells to be photographed, captured as an image, or otherwiseevaluated to determine optical properties can be used in the invention.Typically, a lighting device provides homogeneous illumination to allwells being monitored. A lighting device can also be selected totransmit light of a particular range of wavelengths including, forexample, visible, ultraviolet, infrared, sub-ranges thereof such as red,blue or yellow sub-ranges within the visible range, or combinations ofthese ranges or sub-ranges.

In particular embodiments, the lighting device includes a light emittingdiode (LED) array 36 arranged adjacent the multi-well plate. A cold DCpower source may be utilized to power the LED array. Advantageously, theDC power to each LED or the mechanical position of the various LEOs canbe independently controlled to optimize the imaging of the camera. TheDC/LED configuration of the present invention can be used to minimizeand/or prevents dark or dim images. In particular embodiments, otherforms of lighting such as incandescent or fluorescent lighting can beused. Those skilled in the art will know or be able to determineappropriate illuminating, optical manipulation and imagingconfigurations to provide a sufficiently homogeneous illumination formonitoring a chemical reaction occurring in a plurality of wells.

As shown in FIG. 2, two opposing LED arrays can be provided on eitherside of an aperture or rectangular plate glass window 37, which windowprovides a view of the multi-well plate beneath the glass window. In oneembodiment, one LED array may be positioned slightly higher than theother. Typically, the LED arrays are positioned out of field of view ofthe camera.

One should appreciate that the LED array may vary in accordance with thepresent invention. For example, one, two, three or more LED arrays maybe used. Furthermore, the LED arrays may be configured to utilizedifferent wavelengths, diffusion patterns, voltages, etc. in order toinfluence the imaging of the camera, for example, improving theusefulness of data captured for a particular reaction being monitored.

Other lighting devices that can be used include, for example, one ormore lamps, lasers or bulbs. Such devices are typically selected toprovide homogeneous illumination to a plurality of wells beingmonitored. However, in embodiments in which the light source itself isnot homogeneous a diffuser module can be used to provide homogenouslighting conditions to wells being monitored. One or more lenses canalso be used in combination to provide homogeneous illumination.

If desired the wavelength of light that contacts wells to be monitoredcan be provided by a particular combination of light source and opticalfilters. An optical filter useful in the invention can be any device forselectively passing or rejecting passage of radiation in a wavelength,polarization or frequency dependent manner. Exemplary filters include aninterference filter in which multiple layers of dielectric materialspass or reflect radiation according to constructive or destructiveinterference between reflections from the various layers. Interferencefilters are also referred to in the art as dichroic filters, ordielectric filters. The term can include an absorptive filter whichprevents passage of radiation having a selective wavelength orwavelength range by absorption. Absorptive filters include, for example,colored glass or liquid. Those skilled in the art will know or be ableto determine an appropriate combination of light source and opticalfilters to produce illumination in a desired wavelength range.

Beam splitters, lenses, or other devices for changing the path ofillumination can be used in an apparatus of the invention. Beamsplitters, lenses and their properties are known in the art and can beobtained from commercial sources including, for example, Melles Griot(Irvine, Calif.), or Oriel Corp. (Stratford, Conn.). In particularembodiments, a beam splitter can be used for perpendicular illuminationand viewing a multi-well plate or other plurality of wells. Thus,optical components can be arranged differently from the arrangementsexemplified herein while providing illumination properties similar tothose described herein. Accordingly, an apparatus of the invention canbe configured for compact or modular placement suitable for a variety oflaboratory environments.

If desired, a mask can be used in an apparatus of the invention topreferentially illuminate one or more wells in a multi-well plate orother plurality of vessels. For example, a mask can be placed toselectively illuminate wells of a multi-well plate compared to regionsbetween the wells to prevent unwanted light scatter or to enhancecontrast of images and ease registration of images obtained from themulti-well plate.

In particular embodiments, the intensity of each LED in an LED array canbe independently controlled. For example, each LED intensity to becontrolled by a manual adjustment such as adjustment with apotentiometer. Such control over the LED intensity can also be achievedprogrammatically, for example, via a networked GUI or computer system.An advantage of computer controlled illuminator electronics is thatlighting intensity for all or part of the LED array can be changedremotely via software. Thus, an individual user can interact with a GUIto send commands that alter intensity of one or more LED in an array. Afurther advantage is that illumination intensity can be controlled by afeedback loop in which one or more properties of the illumination aremonitored, automatically compared by a computer algorithm to expectedranges for the one or more properties and intensity of one or more LEOsautomatically adjusted in accordance with commands sent by the computeralgorithm. Thus, a user need not intervene and properties such as LEDintensity, homogeneity of the field of light or color of illuminationcan be adjusted in an automated fashion. Such properties can bedetermined using an image analyzer such as those described below infurther detail.

In one embodiment, camera device 32 is a charge couple device (CCO)camera of the type including, but not limited to, the AXIS 2100 NetworkCamera provided by AXIS Communications, Inc. of Lund Sweden. One shouldappreciate that other types of cameras may be used in accordance withthe present invention. A camera used in the invention can be any devicethat converts a detectable optical property into a signal in a locationdependent manner. Thus, exemplary cameras useful in the inventioninclude, for example, complementary metal oxide semiconductor (CMOS)camera, video camera, internet camera, and other imaging devices capableof converting a picture into a digital image. A camera device of theinvention can be functionally connected to a computer such that adigital image or other data indicative of a signal detected by thecamera can be communicated to the computer for further storage orprocessing or both. The camera device can include a built-inmicroprocessor for example, in one embodiment, the camera includes abuilt-in LINUX device and web server. Such configuration allows thecamera device to be connected directly to the central network and beconfigured to store images in a designated image location on the centralnetwork. Camera modules discussed here incorporate imaging optics,photon detection devices, digitizing electronics, and electroniccommunication capability. These tasks can also be accomplished withseparate components as opposed to an integrated camera module.

A camera device can be positioned on a chemical synthesizer over amulti-well plate or other plurality of wells such that it is capable ofobtaining an image which includes all of the wells, as is shown in FIG.1 for an oligonucleotide synthesizer. The multi-well plate generallyincludes 96 or 384 wells, however, one should appreciate that themulti-well plate may include more than 384 wells. Advantageously, thecamera device not only allows observation of the multi-well plate as awhole, but also allows observation of each well individually. A cameradevice can incorporate an optical filtering stage rendering the detectorcapable of spectral measurements and tunable to specific chemicals thatexhibit specific absorptivities in spectrum. Exemplary optical filtersthat can be used to filter light entering the camera include, withoutlimitation, those set forth above with respect to lighting devices. Anoptical filter used for a camera device can match the emission of aparticular lighting device in order to create a powerful absorptionmeasurement in reflection mode and for selective detection of desiredsignals in a well to be detected or selective rejection of unwantedbackground signals. An RGB (Red, Green, Blue) camera device can also beused instead of a single color device. As with other cameras describedherein, an RGB camera can be used without an optical filter if desired.

A camera device useful in the invention can include an internal cameramicroprocessor having an image acquisition program 38 that can besignaled to take an image and transfer the image electronically to astorage location such as a central network file repository or imagestorage location 39 on the central network shown in FIG. 3. The imagestorage location can be a path location on the network. The imagestorage location is not machine specific and may be provided ondifferent platforms including, but not limited to LINUX®, WINDOWS®, andother suitable operating systems such as those set forth hereinpreviously. The storage location can be dynamically configurable by eachof the chemical reaction monitor's components, so if a problem occursall subcomponents of the software component can be pointed to adifferent location with minimal effort.

In the exemplary embodiment shown in FIG. 3, the oligonucleotidesynthesizer system is configured to control the camera device. Forexample, OLIGATOR® control software may be configured to signal thecamera device, via the central network, when to take an image of amulti-well plate of the oligonucleotide synthesizer. The image may betransferred to the image storage location (see FIG. 3) in the form of aJPEG, TIFF, BMP file or other suitable file format.

In particular embodiments, the image files are named according to batchnumber, plate number, cycle number and loop number within the synthesisprotocol that the synthesis process is at when the image is taken. Inthis respect, the term “cycle” refers to the complete cycle of steps forthe addition of each base to the growing DNA chain, including thedeprotection, coupling, capping, and oxidation steps. The term “loop”refers to an operation that is performed several times within the samecycle. For example, the deprotection step may repeat two or three timeswithin the same cycle whereby a “loop” designation may be used todifferentiate the particular deprotection operation within a cycle.

Turning now to FIG. 3, which figure illustrates an exemplary softwarecomponent of chemical reaction monitor 30 in accordance with the presentinvention, separate software subcomponents or programs can be providedto independently or autonomously store, analyze, and view the images.For example, the camera device may include means such as imageacquisition program 38 to autonomously send images to image storagelocation 39 on the central network. Similarly, the various subcomponentsincluding, but not limited to, the broker, analyzer, viewer, multipleanalogue and/or real-time data management programs described below maybe separate and discrete from one another in order to removedependencies. The network architecture of FIG. 3 is exemplary and thoseskilled in the art will recognize that a variety of other architecturesand modifications of the exemplified architecture can be used to producea chemical reaction monitoring system having the properties set forthherein.

An autonomous computer system configuration provides independence thatallows images to be obtained and/or viewed without overburdeningresources necessary for image analysis. This also allows individualdevelopment of the different subcomponents in different languages and/orplatforms, by different engineers. Such autonomy also allows anyone ofthe many subcomponents to be taken offline, causing at most, the othersubcomponents to patiently wait for the off-line subcomponent to returnto service, as opposed to a major system shut down. Thus, if one of theimaging, analyzing, or viewing subcomponents malfunction, themalfunction will not affect the remaining programs. For example, if theviewer program crashes, the discrete camera imaging software stillallows the camera to continue capturing and storing images, which imagesmay continue to be analyzed and, once the viewer program is againrunning, allow one to again view the stored images.

In one embodiment, when a synthesis starts, and in particular, when adeprotection step is encountered, an image is taken of the multi-wellplate (see FIG. 6) and sent to the image storage location. The imagestorage location may be local or it can reside at a particularcentralized network address. A broker program 40 can be configured toconstantly observe the image storage location for new images. Forexample, the broker program can constantly observe the image storagelocation and define images as being “new” merely by their presence inthis location, and by having a specific file extension, for example“.jpg” in the case of JPEG images, and/or other suitable image formatextensions. The broker program can build a list of new image file namesevery time the queue is empty.

A broker program, replete with an internal queue of unique imagefilenames, can reside at an IP address on a network and can assign aunique file name to each new image. A filename can encode informationthat is specific to a particular synthesis and step of the synthesis,such as the plate, the batch, the cycle, loop, machine name, etc.

A separate analyzer program 41 can be responsible for processing storedimages. The analyzer program can run in the background autonomously andindependent of the image storage program and other programs of thesoftware component.

The analyzer program can scan for fresh images, that is, unprocessed orto-be-analyzed images, through a broker program. Once the analyzerprogram gets the filename of a fresh image, it can retrieve the filefrom the network location and perform image analysis. The analyzerprogram can individually open each image stored in the image storagelocation (see FIG. 3), register the image geometrically, then samplespecific regions of the image for specific values that relate to themagnitude of the hue, saturation and luminance of the bright orangecolor which is emitted as DMT is released during the deprotection stepof oligonucleotide synthesis. The specific regions of the image sampledcan correspond to each well of the multi-well plate that has been imagedor photographed. Typically, the specific regions correspond to thecenter of each well. However, if desired any part of each well can becorrelated with specific regions of the image.

The analyzer program can analyzes one or more optical property, such asDMT intensity, for every well in a multi-well plate, extract theintensity value and store the intensity value in memory. For example,the specific values which relate to the magnitude of the hue, saturationand luminance of each well of the image can determined and stored. Oncein memory, the values for a particular optical property can be formedinto a structured query language (SQL) statement and appended to an SQLdatabase or analysis results database. After the database update iscomplete, the analyzer program can move the file to a new “processed”location and changes the extension of the filename, for example, from“.jpg” to “jpr’ to indicate in a human-readable form that the image hasindeed been processed.

At this point, the analyzer program may request another image from thequeue. The analysis or processing cycle for each image typically takesless than approximately 10 to 20 seconds and can be about 8 seconds orless. The chemical reaction monitor may be provided with multiple copiesof the analyzer program, running from different personal computersthroughout a company's network. For example, 1, 2, 3 or more analyzerprograms may simultaneously connect to the network and simultaneouslyanalyze different images. Thus, as workload (e.g., the number of imagesto be analyzed) increases, additional analyzer programs can be startedto ensure that the rate of analysis meets or exceeds a desired rate ofimage generation to ensure that throughput demands are met.

In some instances, the synthesizer requires the use of multi-well plateshaving filters or frits 42 (see FIG. 6). It has been determined thatfrits have a time varying intensity that is not linearly proportional toDMT intensity or concentration. It is hypothesized that this is due tothe individual absorption rates of each frit, and drain rate of eachwell. In the event that frits are utilized, the frit is generallyclearly visible in the bottom of each well. The analyzer program can beconfigured to programmatically exclude the image of the frit from datacollection. Similarly other features present in an image of a pluralityof wells can be programmatically excluded from data collection.

Continuing with the exemplary oligonucleotide synthesizer shown in thefigures, the analyzed images can be stored elsewhere on the centralnetwork (e.g., with the “jpr’ extension) and specific values whichrelate to the magnitude of the hue, saturation, luminance or thegrayscale intensity of each well of the image can then be logged into ananalysis results database 43 on the central network (see FIG. 3). Thisdatabase can then be queried for either “real-time” monitoring and/or“historical” monitoring. Such configuration allows a technician to querythe analysis results database while a process is going and visuallyidentify problems in a near real-time fashion. Such configuration alsoallows future review of the storage location in a quality control (QC)fashion to look at images of completed batches for confirmation ofproblems that have happened in the past (i.e., historical monitoring)during the completed batches. The data analysis storage location cancontain two table structures, one for laboratory information managementsystem (LIMS) sequence data and one for real-time DMT values. Thestorage location may be comprised of any suitable database softwareincluding, but not limited to MICROSOFT® SQL SERVER® and MICROSOFTACCESS®.

Certain determinations about the synthesis may be made from the observedimages and/or the stored data. For example, the degree of colordegradation may be tied to coupling efficiency in which a lower degreeof color degradation indicates a higher coupling efficiency.

Observing a significant decline in the magnitude of the orange coloremitted by the release of DMT can provide an early indication that asynthesis is suffering a general or catastrophic failure. In thisregard, relatively quick color degradation within a couple of cycles isindicative of failure. Observation of such color degradation allows thesynthesis to be stopped, automatically or by a technician, and restartedwith fresh materials, thus saving time and money. Alternatively thesystem can be configured to automatically stop synthesis when changes inoptical characteristics deviate substantially from a predefinedthreshold or range of acceptable values.

Individual wells or groups of wells that lose color can indicate aclogged nozzle in either a base or bulk delivery nozzle array of asynthesizer. For example, if a loss of color is observed in the samewell of all plates, the color loss may indicate that a bulk reagentnozzle used in delivering reagents to multiple plates is clogged.Similarly, if a loss of color is observed in an entire row of a singleplate, the color loss may indicate that a dispenser which should bedelivering regents to the row is clogged.

The analyzer program can have access to the source sequence or protocolfile for polymers to be synthesized in order to avoid confusing a failedindividual polymer with one that is completed. The architecture of thissoftware can be such that an independent viewer program 44 is providedto allow a technician to monitor the synthesis in near real-time that,in turn, allows for subjective evaluation apart from the analyzerprogram.

In one embodiment, a chemical reaction monitoring device of theinvention is provided with a multiple algorithm detector (MAD) program45. The MAD program obtains a list of current batches that are beingsynthesized and uses these for analysis as follows. Once the list isestablished, the MAD program retrieves all of the DMT data for each welland each plate for an entire synthesis batch while the synthesis isoccurring. The MAD program then works its way through all of the wellsand all of the plates for that batch, examining the data for each welland rendering a pass/fail decision of the chemical reaction within thatwell. The pass/fail decision is written back to the results analysisstorage location. The list of batches is re-evaluated every few minutesto update this list and the pass/fail analysis repeated for the newlist.

In some processes, oligonucleotide synthesis may have a protocol thatcontains N-number of deprotection loops (n being an integer value of oneor more), in which case, such information is stored in a protocol file.The MAD program of the present invention may be configured toautonomously determine the number of deprotection loops. In this regard,the MAD program determines the number of deprotection loops based on theimage file names. As noted above, the cycle, loop, and other informationcan be encoded in to the image filename. Thusly, the MAD program maycalculate the summation of the data for all of the loops (1, 2, 3, . . .N number of loops per cycle). Similar to the analyzer program describedabove, the MAD program can take about 10 to 20 seconds, and, inparticular embodiments can take about 8 seconds or less, to analyze thedata for an entire synthesis. The MAD program can continuously performanalyses as new data is constantly being added to the analysis resultsstorage location.

Though the structure of the MAD program can support N-number of failuredetection algorithms, a primary algorithm in the MAD program is anexponential fit algorithm. For example in the case of oligonucleotidesynthesis using DMT deprotection steps, it is known and has beenobserved that DMT intensity decreases at a predictable rate. In general,this rate is clearly observable given typical molar concentrations ofDMT and the range of cycles. DMT intensity “slightly” or “gently”decreases as synthesis progresses and has been found to decay in anegative exponential decay according to the following equation:

DMT Value=e ^(−kx)  Eq. 1

where the value of “k” is the experimentally determined extinctioncoefficient which is a function of coupling efficiency and extinctioncoefficient that dictates the rate or degree in which the DMTdiminishes, and “x” represents the cycle of the particular data point.

The MAD program may be configured to detect synthetic failures bycalculating an “expected” optical signal for each well at any givencycle and comparing a detected value to the expected value. Detectedvalues that differ substantially from the expected value are indicativeof failure, whereas detected values that are sufficiently similar to theexpected value are indicative of a passing score. For example, in thecase of oligonucleotide synthesis, an expected DMT intensity value canbe calculated based on the initial deprotection values and theparticular cycle. The expected value is determined by a function derivedwhere the expected DMT value (EV) is a function of initial reading,coupling efficiency, and cycle as follows:

EV=I _(o) *e ^(−(100-eff)/100*C)  Eq. 2

where “I_(o)” is the average initial intensity of the first threevalues, “eff” is the desired or assumed coupling efficiency in percentand “C” is the particular cycle expressed in an integer value from 0 to100. During operation, the MAD program continuously compares the currentDMT value against the expected value. Once the difference between theexpected and real DMT value exceeds a predetermined threshold, the wellor oligo is flagged as “failed”. For example, FIG. 9 illustrates DMTvalues for 1st, 2nd and 3rd deprotection loops of a particular wellwithin a oligonucleotide synthesis cycle. In particular, there are threeDMT loop values (y-axis) for every cycle (x-axis) and trityl value forthe z-axis. The rear row represents an expected DMT value. In this case,it is apparent that the oligonucleotide suffered a catastrophic eventaround cycle no. 3 as the DMT values of the third cycle deviatesubstantially with respect to the expected value. In turn, the well canbe flagged as “failed”.

Acceptable values for a passing or failing synthetic step can beselected based on a tolerance level chosen by a particular user. Forexample, in the case of a relatively short polymer a relatively highloss in yield can be tolerated including, for example, a loss resultingin at least 85%, 90%, 91%, 92%, 93% or 94% yield per step. However, forsynthesis of longer polymers lower loss of yield per step is typicallytolerated since total yield loss is additive. In such cases a tolerablestep yield can be, for example, at least about 95%, 96%, 97%, 98%, 99%or 99.5% yield per step. The pre-determined threshold may be set veryconservatively such that exceeding its limits all but ensures theoligonucleotide within the particular well contains too many failureproducts for use. One should appreciate, however, that other algorithmsmay be provided to perform more detailed analysis, corroborate withknown standards, and/or define new standards.

As illustrated in FIG. 1, a graphical user interface (GUI) such as aflat-panel monitor or touch screen 46, a personal computer (PC) or othersuitable means, may be provided on the oligonucleotide synthesizer tofacilitate near realtime monitoring in which the GUI provides auser-friendly software interface on the oligonucleotide synthesizer.This configuration allows a technician to access the viewer program andthus view an image of the chemical reaction within the wells of themulti-well plate while the oligonucleotide synthesizer is running. Suchconfiguration also allows a technician to browse the latest images (see,e.g., Photograph—Plate 4 in FIG. 6) and thus provides a near real-timemonitoring configuration. Accordingly, the technician can be notified ofa detected failure in a substantially real-time manner such that theuser can stop the synthesis if desired.

In one embodiment, the viewer program is a graphical user interface(GUI) program, which is provided to readily provide user-friendly datato a technician or other authorized personnel. The GUI program providesa component of the chemical reaction monitoring system that is directlyused by humans allowing human interaction with the system. Accordingly,the system can communicate information to a user through the GUI.Conversely, a user can provide information or a command to the systemthrough the GUI program. The GUI program may connect to the analysisresults storage location and may continuously retrieve the latest set ofanalyzed data. The GUI program can be configured to output a visualrepresentation of the stored data. In one embodiment, the GUI programvisually provides the user information regarding synthesis by way oftouch screen 46 or other suitable means. The GUI program also serves ascentralized real-time nerve center that allows anyone on a PC connectedto the network to use, review or manipulate data in connection withongoing or previously performed chemical reactions.

In one embodiment, the GUI program provides a windows-type graphicalinterface that may include a digital photograph of a specified plate orother representation of a plate. The GUI may also provide a visualrepresentation as to the status of the chemical reactions taking placewithin each well. For example, wells that are marked as “failed” may beshown in red (e.g., vertical crosshatching of wells A2 through A8 andA10), while wells that marked as “pass” may be shown in green (e.g.,angled cross-hatching of wells A1, A9, A11 and A12). The GUI program maygraphically provide the DMT intensity values of each well of amulti-well plate as shown in the lower left-hand corner of FIG. 6,and/or the cumulative DMT intensity values within a specified well, asshown in the upper right-hand corner of FIG. 6. One will appreciate,however, that the GUI program may be configured to visually representthe data of the analysis results storage location in other suitablemeans including, but not limited to, numeric representations. graphs,charts and/or tables.

In one embodiment, the chemical reaction monitor of the presentinvention is configured for real-time data monitoring (RTDM). A RTDMprogram 47 may be provided independently of, or integral to, thegraphical user interface program or other program of the invention. TheRTDM program mines data, such as that described above, for patterns offailure and renders a “pass/fail” decision on whole or partialprocesses.

The typical cycle time of an oligo coupling, one step of anoligonucleotide synthesis, takes approximately 10 to 15 minutes. Asnoted above, the time of a complete analysis or processing cycle of theanalyzer program and the MAD program may be approximately 8 seconds orless. As the processing cycle represents a small percentage time ascompared to the oligo coupling (e.g., 0.8% to 1.3%), the chemicalreaction monitor of the present invention may be considered “real-time”.Furthermore, the rate of analysis or processing by the analyzer and MADprograms may be substantially equal to or greater than the rate of imagegeneration or acquisition by the camera. In particular, the analyzerprogram may process an image and export the data extracted from theimage to the analysis results storage location substantially as fast asan image is created. In other words, as soon as a picture is taken, theanalyzer program analyzes the image and the data is logged.

Given that the data is available from the analysis results storagelocation in “real-time”, the RTDM program can be configured to occurcontinuously. In particular, the RTDM program may serve as aquality-control program that continuously scans the analysis resultsstorage location for new records and performs data mining from thestored data to ascertain whether or not a particular chemical reactionhas passed or failed quality control.

In the case of oligonucleotide synthesis, there are many differentfailure patterns that one may observe in DMT intensity. For example, theintensity, or “concentration” of DMT directly correlates to the amountof and the quality of the oligonucleotide. In general, for a givenoligonucleotide within a well, if the same DMT level is maintainedthroughout the entire synthesis, it is generally accepted that thequality of the oligonucleotide will be acceptable (see e.g., FIG. 4). Onthe other hand, should any significant changes be observed in the DMTlevel, such as an increasingly negative slope, or an exponential decay,etc, the oligonucleotide quality would likely be unacceptable (see e.g.,FIG. 5).

FIG. 4 shows a graphical representation of DMT intensity measurements ofa well of a multi-well plate taken during each cycle, and loop thereof,during an exemplary successful oligonucleotide synthesis. The rear rowrepresents the final or summed measurement of all of the discrete DMTmeasurements that have occurred throughout the synthesis. In thisexample, the rear row “approximates” a straight line indicating that theDMT level is substantially maintained throughout the entire synthesis.The rear row does not significantly slope up or down, nor does the rearrow have any aberrations. Instead, the substantially flat graphindicates that the oligonucleotide in question is “good”.

In contrast, FIG. 5 shows a graphical representation of DMT measurementsof a well of a multi-well plate taken during an exemplary unsuccessfuloligonucleotide synthesis. The DMT intensity drops off sharply aroundcycle 9. In addition, the rear row, which again represents the final orsummed measurement, resembles the unit step function indicating that thelevel of DMT intensity as significantly decreased. As opposed to the“flat” response of the “good” oligonucleotide in FIG. 4, the stepresponse of FIG. 5 indicates that the oligonucleotide is “bad”.Accordingly, a failed polymer synthesis can be identified due to adeparture from an expected trend in a plot of optical signal change vs.cycle number.

As the DMT intensity can be monitored to determine whether anoligonucleotide is “good” or “bad”, an RTDM program may be configured tomine the data of an analysis results storage location for patternssimilar to those described above, alerting the appropriate personnelwhen failures are detected.

Advantageously, the chemical reaction monitor of the present inventionallows parallel observation of a plurality of wells and allowsobservation of each well individually. Furthermore, the monitor of thepresent invention allows monitoring a plurality of wells with oneimaging device.

Advantageously, the chemical reaction monitor of the present inventionallows capturing data, analyzing the data, and using the data in a nearreal-time manner or in a historical manner to measure performance of theoligonucleotide synthesizer. The monitor is configured to detect andannounce suspected failures of oligonucleotides, and may be configuredto render a quantifiable number relating to oligonucleotide quality,akin to coupling efficiency and so on.

Advantageously, the imaging, analyzing, and viewing processes can bediscrete and autonomous. If anyone or more of the processes crashes orotherwise fails, the chemical reaction monitor of the present inventioncan render a diagnosis based on the nature of the failure andcommunicate one or more potential solutions to a graphical userinterface. The monitor can further interrupt one or more steps of asynthetic reaction. In one embodiment synthesis in all wells can bepaused until a further command is provided by an external user. In otherembodiments, delivery of reagent to a subset of wells that haveexperienced a failure can be discontinued. An advantage of the monitoris that interruption of only a subset of synthetic steps, such assuspending reagent delivery to a subset of failed wells, allows theremaining processes to continue operation while avoiding wastefuldelivery of reagents to previously failed wells. Furthermore, the systemcan be configured such that if the analyzer program malfunctioned, thechemical reaction monitor of the present invention would allow theimaging and viewing processes to continue.

A chemical reaction monitor of the present invention can be configuredto make decisions regarding whether or not to continue synthesis on amulti-well plate based on one or more criteria including, withoutlimitation, the cost of proceeding with the synthesis vs. the value ofthe synthesis, time constraints on instrument scheduling based on thenumber of plates to be processed after the particular plate beingmonitored, the state of repair of the machine based on identification ofglobal failures as well as other quality metrics determined in amanufacturing environment. Information regarding the optical propertiesof a reaction can be collected, displayed or manipulated in the timedomain (i.e. intensity vs. time). In particular embodiments, time domaindata can be converted by Fourier transform to provide data in thefrequency domain. Thus, periodicity in optical property data will resultin an observable frequency component. The Fourier transform can becoupled with wavelet analysis such that the frequencies can becorrelated with different variables, such as the particular monomerbeing reacted in a given cycle and well (for example, an A, C, G, or Tphosphoramidite monomer), the particular instrument being used (in caseswhere multiple instruments are being monitored) or a particularsubcomponent of an instrument (for example, a valve or delivery nozzle).Wavelet analysis incorporates a third dimension of the transform so thatsuch variables can be represented in the third dimension and evaluatedto determine if criteria important to a manufacturing environment arebeing properly met. If criteria are not being met the information fromwavelet analysis can be used to direct adjustment of instrument activityto achieve a desired manufacturing goal. Such adjustments can becommunicated to a GUI in the form of suggested solutions to a perceivedmalfunction. Alternatively, adjustments can be made in an automatedfashion using a feedback loop in which the function of a component ofthe instrument is automatically altered in response to the perceivedmalfunction.

In one embodiment, the chemical reaction monitor includes a dataregistration and extraction algorithm for determining the DMT intensitywithin each well, which algorithm may include one or more of thefollowing steps:

1) Read image;

-   -   a) Determine plate configuration (e.g., 96 or 384 well plate);

2) Register;

-   -   a) Use fiducial mark on plate holder (384 wells);        -   i) Move to middle top well and pattern recognize;        -   ii) Move to left and right of top row and recognize wells;        -   iii) Create fit of line of centers of top row wells;        -   iv) Use to calculate angle of rotation;    -   b) OR use 2 corners of plate well detection (96 and/or 384 well        plates);        -   i) Use to calculate angle of rotation;    -   c) Rotate image;

3) Find frit location;

-   -   a) Find all well centers using plate measurements;    -   b) Apply algorithm of optical parallax to identify frit position        with respect to well center as viewed by camera;        -   i) OR use 2×2 algorithm and thresholding to pattern            recognize frit features independent of well positions on            plate;    -   c) Create circle around frit with known diameter;    -   d) Create circle around center of well with known diameter;    -   e) Average intensity of pixels inside outer circle but w/out        frit area;

4) Store values in analysis results database per plate per cycle perwell per run;

5) Repeat above for new image automatically.

In one embodiment, the chemical reaction monitor includes a dataanalysis and Pass/Fail algorithm for identifying specific failedoligonucleotides, which algorithm may include one or more of thefollowing steps:

-   -   1) Read image data per loop per plate per synthesis cycle per        well from analysis results database;    -   2) Read info for subject oligonucleotide from LIMS database        (e.g., sequence length);    -   3) Sum all 3 loops per synthesis cycle (see, e.g., FIG. 4);    -   4) Determine average Intensity of first 3 cycles for each well;        -   a) If no value, assume no data and then set initial            Intensity value accordingly for monitor cameras from            configuration file;        -   b) If low value, FAIL oligonucleotide;    -   5) Check last synthesis step for modifier from LIMS;        -   a) If modifier does not have DMT then exclude last cycle            from trending;    -   6) Determine Intensity exponential decay vs. cycle index;        -   a) Assume coupling efficiency at 98.5%;        -   b) Calculate expected value of DMT for each cycle based on            assumed coupling efficiency;        -   c) Assume allowed variation of calculated vs. measured DMT            value at set % value form configuration file;        -   d) Compare summed loop value with calculated value for every            cycle;        -   e) If difference>+−se value then FAIL oligonucleotide;    -   7) Determine scaling of DMT intensity between loops;        -   a) Loop1/loop2/loop3 Intensity ratio for all cycles;        -   b) Compare with set criteria;        -   c) If outside set criteria then FAIL oligonucleotide;    -   8) Step detection in synthesis;        -   a) Check all loops and sum of loops for appearance of step            (decrease or increase) in DMT intensity;        -   b) Compare step with set value from configuration file;        -   c) If step>set value FAIL oligonucleotide;    -   9) Store PASS/FAIL values in database per plate per cycle per        well per run;    -   10) Repeat above for new image automatically.

In one embodiment, the chemical reaction monitor includes a further dataanalysis and Pass/Fail algorithm for identifying trends for improvingquality control and quality assurance, which algorithm may include oneor more of the following steps:

-   -   1) Read Pass/Fail result per loop per plate per synthesis cycle        per well from database;    -   2) Read info for this oligonucleotide from LIMS database, e.g.        sequence length;    -   3) Pattern recognize Row-wise or Column-wise global failures;    -   4) Pattern recognize local area failures;        -   a) If concentration of FAILED oligonucleotides within            specific area of plate;    -   5) Compare with failure mode from configuration file;    -   6) Alert Real-Time data monitoring for failure mode;    -   7) Pause oligonucleotide synthesis;    -   8) Send Failure mode result to LIMS database;    -   9) Repeat above for all runs.

In one embodiment, the chemical reaction monitor includes a furtheralgorithm for monitor and control synthesis, which algorithm may includeone or more of the following steps:

-   -   1) Get Pass/Fail per well per oligonucleotide;        -   a) Calculate from previous algorithm submitted;        -   b) Receive data form database real time using RTDM GUI            monitor for specific synthesis batch;    -   2) According to pass/fail criterion for whole plate and/or whole        synthesis batch and using the above P/F numbers for individual        wells, decide on plate/batch quality;    -   3) Alarm operators on quality;        -   a) Use % number of failed/good wells in plate/batch;        -   b) Use a “score” of goodness for plate/batch calculated            using above criterion;    -   4) Proceed to pause only synthesis;    -   5) Suggest different modes/action plans of hardware        configuration for rest of synthesis cycles;        -   a) E.g. not firing all banks;        -   b) E.g. resynthesizing only one of plates in-situ in            synthesizer system;        -   c) Etc.;    -   6) Operator confirms actions suggested form menu;        -   a) Possibly, for specific, already confirmed or emergency            modes, operator input is bypassed and action is taken by            computer on stopping/continuing synthesis in specific mode;    -   7) Action taken and control of synthesizer system is performed        by software component of monitor or via native software of        synthesizer system;    -   8) Synthesizer system returns to normal operation with        monitoring only; Parallel, Return log and values of actions        taken above in analysis results database.

In another embodiment of the present invention, chemical reactionmonitor 30 a is similar to chemical reaction monitor 30 above butincludes a modified lighting device 33 a and a modified softwarecomponent. Like reference numerals have been used to describe likecomponents of 30 and 30 a. In this embodiment, the lighting deviceincludes a single LED array 36 a pivotally mounted and positioned to oneside of glass window 37 a, as shown in FIG. 10. In this embodiment, atransparent reflector 48 is provided which directs the light rays of theLED array downward to illuminate multi-well plate 34 a. The transparentreflector allows camera device 32 a to take a picture of the multi-wellplate when the LED array is pivoted downward and in place about theglass window illuminating the multi-well plate. A diffuser or opticalfilter or both can be applied prior to the light hitting the transparentreflector (beam splitter) if desired, as set forth previously herein.

In this embodiment, the software component includes an integrated viewerprogram and RTDM program, generally referenced by the numeral 49 in FIG.12. One will appreciate that any two or more of the softwaresubcomponents may be integrated into a single program. Thus, theprograms and network locations set forth above are exemplary and notintended to limit the number or type of functions that can be performedby particular components or programs.

For convenience in explanation and accurate definition in the appendedclaims, the terms “up” or “upper”, “down” or “lower”, “left” and “right”are used to describe features of the present invention with reference tothe positions of such features as displayed in the figures.

In many respects the modifications of the various figures resemble thoseof preceding modifications and the same reference numerals followed bysubscript “a” designate corresponding parts.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the Claims appended hereto and theirequivalents.

1. A method for synthesizing a plurality of polymers comprising: (a)providing a plurality of wells, each well comprising a polymer; (b)dispensing a reagent comprising a reaction component to said pluralityof wells under conditions sufficient for said reaction component to beincorporated into said polymer; (c) obtaining an image of said pluralityof wells and saving said image data to computer memory; (d) accessingsaid image data in computer memory and determining a specific valuecorresponding to the extent of chemical reaction within each well at thetime the image was obtained; (e) determining whether a differencebetween a value expected if the chemical reaction is successful and thespecific value indicates failure of the chemical reaction within a well;and (f) repeating steps (a) through (e), wherein said dispensing is toat least one well where failure of the chemical reaction is notindicated, while discontinuing dispensing to one or more wells wherefailure of the chemical reaction is indicated.
 2. The method of claim 1,wherein step (e) further comprises communicating a representation ofsaid specific values to a graphical user interface.
 3. The method ofclaim 2, wherein step (f) further comprises receiving a command fromsaid graphical user interface to repeat steps (a) through (e).
 4. Themethod of claim 1, wherein step (c) further comprises removing saidreagent from said plurality of wells after obtaining said image.
 5. Themethod of claim 4, wherein removing said reagent comprises acentrifugation or aspiration.
 6. The method of claim 1, wherein saidpolymer comprises a polynucleotide.
 7. The method of claim 1, whereinsaid plurality of wells comprises a multi-well plate.
 8. The method ofclaim 1, wherein step (c) further comprises illuminating said pluralityof wells with a light emitting diode (LED) array.
 9. The method of claim1, wherein said image is obtained with a charge couple device (CCD)capable of imaging the plurality of wells simultaneously.
 10. A methodfor synthesizing a plurality of polymers comprising: (a) providing asubstrate comprising a plurality of fixed locations, each locationcomprising a polymer associated therewith; (b) dispensing a reagentcomprising a reaction component to said plurality of locations underconditions sufficient for incorporation of said reaction component intosaid polymer; (c) obtaining an image of said plurality of locations; (d)determining a specific value corresponding to the extent ofincorporation of the reaction component into the polymer at eachlocation at the time the image was obtained; (e) determining whether adifference between a value expected if incorporation of a reactioncomponent into the polymer is successful and said specific valueindicates failure of the polymer synthesis at a location; and (f)repeating steps (a) to (e), wherein said dispensing is to locationswhere failure of the incorporation of a reaction component into thepolymer is not indicated, while discontinuing said dispensing to one ormore locations where failure of the incorporation of a reactioncomponent into the polymer is indicated
 11. The method of claim 10,wherein step (f) further comprises resuming said dispensing to said oneor more locations if one or more criteria are met.
 12. The method ofclaim 10, wherein step (f) further comprises resuming said dispensing tosaid one or more locations if the value of the synthesis is greater thanthe cost of the synthesis.
 13. The method of claim 10, wherein thereaction component is selected from the group consisting of monomericreaction components and polymeric reaction components.
 14. The method ofclaim 10, wherein the synthesis comprises polynucleotide synthesis. 15.The method of claim 14, wherein the specific value is determined bydetermining the amount of a blocking group released from the polymer.16. The method of claim 15, wherein the blocking group comprisesdimethyltrityl.
 17. The method of claim 10, wherein the plurality offixed locations comprises a plurality of wells.
 18. The method of claim10, wherein the substrate comprises a multiwell plate.
 19. The method ofclaim 10, wherein the one or more locations comprises a subset oflocations where failure is indicated.
 20. The method of claim 10,wherein the value expected if the incorporation of the reactioncomponent into the polymer is successful and said specific value eachcomprises a plurality of measurements.